The long term objective of this research is to understand the molecular basis of position effects on gene expression. Position-effects refer collectively to instances in which the position at which a gene resides in the genome influences the expression of that gene. This research focuses on the position-dependent repression of mating-type genes in Saccharomyces cerevisiae, as an example of position effects. In this case, the mating type genes at the HML and HMR loci are repressed by the action of the four SIR genes, whereas the mating type genes at MAT are unaffected. Recent evidence indicates that SIR genes may control expression through effects skin to heterochromatic inactivation, as has been described in Drosophila. It is likely that this level of regulation will be a common means in which expression of gene is turned off in all eucaryotes. Indeed, silencer elements that mediate position effects have already been identified in human viruses and adjacent to cellular oncogenes. In addition, a position effect is likely to be the basis of the fragile-X syndrome, the leading genetic cause of mental retardation. Our studies will focus on yeast due to the well developed background on a particular position effect, and because this organism promises to provide the first mechanistic glimpse into how position effects work. The following broad areas will be explored during the next five years: 1) The silencer elements that act in cis to mediate position effects will be studied to define unequivocally which proteins act at the silencer in vivo. The connection between the SIR proteins, required for the position effect, and the proteins that bind the silencer will be tested in vitro. 2) The role of DNA replication in silencer function will be determined. In particular, the role of a protein kinase required both for the position effect and for the initiation of replication. 3) the specificity of the position effect extends to the interactions between a site-specific endonuclease and its recognition site. 4) A specific model for the role of silencers in defining domains of chromatin will be tested in vivo. 5) An in vitro assay of position effects will be sought in order to study the molecular mechanism with purified components.